The invention relates to cyclic peptides and to molecular tube structures constructed from cyclic peptides. More particularly, the invention relates to the use of cyclic peptides having amino acid sequences with a repeating D-L-chirality motif employable for constructing self-assembling molecular tubes.
Cyclic peptides form a large class of natural and synthetic compounds. Naturally occurring cyclic peptides have diverse biological activities, e.g., antibiotics, toxins, hormones, and ion transport regulators. Naturally occurring cyclic peptides are not known to be synthesized via mRNA transcription, i.e., the amino acid sequence of naturally occurring cyclic peptides is not coded by the genome of the organism producing the material. Instead, the synthesis of naturally occurring cyclic peptides is dependent upon a series of non-transcriptional enzymes specifically dedicated to the synthesis of these products. Many cyclic peptides employ both amide and non-amide linkages and incorporate unnatural amino acids, i.e., amino acids not utilized in the mRNA transcriptional synthesis of linear proteins. Both D- and L-enantiomers of amino acids are widely employed in natural and synthetic amino acids. Synthetic analogs of several naturally occurring cyclic peptides have been designed and synthesized with modified biological activity.
Chemically, cyclic peptides are divided into two categories, i.e., homodetic peptides and heterodetic peptides. Homodetic peptides consist entirely of amino acid residues linked to one another by amide bonds. The present application is directed entirely to cyclic homodetic peptides. Heterodetic peptides include linkages other than amide linkages, e.g., disulfide linkages and ester linkages. Depsipeptides are a type of heterodetic peptide. Depsipeptides employ ester linkages. Valinomycin is a cyclic depsipeptide with an alternating chiral D-D-L-L-motif employing ester linkages within the ring. The present application specifically excludes heterodetic peptides. The chemistry of both homodetic and heterodetic cyclic peptides is extensively reviewed by Ovchinnikov et al. (1992), The Proteins, Vol. V: 307-642.
Molecular tubes are not previously known to be formed by cyclic peptides but are known to be formed by linear peptides. For example, gramicidin A is a linear pentadecapeptide having an alternating chiral D-L-motif. When integrated into a target bio-membrane, gramicidin A forms a left-handed anti-parallel double-stranded helix with 5.6-6.4 amino acid residues per turn. Gramicidin has an average outer diameter of approximately 16 xc3x85 and an average inner diameter of approximately 4.8 xc3x85. The inner channel of gramicidin serves as a path for passive transmembrane ion transport. (See: Wallace, B. A. et al. (1988) Science, 44: 182-187; and Lang, D. (1988) Science, 44: 188-191.)
Molecular tubes may be formed from materials other that amino acids. Carbon tubes are disclosed by Iijima (Nature (1991), 354: 56-58) and Ebbesen et al. (Nature (1992), 358, 220-222). These carbon tubes are composed of graphite and have a concentric close ended structure. Inorganic tubes find wide application in chemistry, e.g., micro- and meso-porous inorganic solids known as zeolites are employed for enhancing a variety of reactions. The area of zeolites is reviewed by Meier et al., Atlas of Zeolite Structure Types, 2nd Edn (Butterworths, London, 1988).
What is needed is a method for assembling and disassembling molecular tubes of varying length and width using interchangeable subunits. What is needed is a versatile subunit for implementing the above method, i.e., a subunit which responds to a undergoes self-assembly and self-disassembly upon. What is needed is homodetic cyclic peptides which can be employed as subunits for self assembling and disassembling molecular tubes.
The invention includes cyclic homodetic peptides employable for assembling and disassembling molecular tubes, molecular tubes assembled from such cyclic homodetic peptides, and methods for assembling and disassembling such molecular tubes.
Cyclic homodetic peptides included within the invention have a stable disk conformation which facilitates the self-assembly of such peptides to form molecular tubes. A stable disk conformation is achieved by designing the cyclic peptides with a repeating D-L-chirality motif. Conformance with this repeating chirality motif necessitates that the amino acid sequence of the cyclic peptide include only an even number of amino acid residues. Since glycine lack chirality, conformance with this repeating chirality motif also necessitates that the amino acid sequence of the cyclic peptide exclude glycine or minimize the inclusion of glycine.
A stable disk conformation is further favored by limiting the size of the cyclic peptide, viz., the amino acid sequence of the cyclic peptide includes between 6 and 16 amino acid residues total. The stability of the disk conformation of cyclic peptides tends to decline with increasing ring size due to statistical mechanics considerations. Cyclic peptides with ring sizes greater than 16 residues are less preferred due to the low stability of their disk conformation.
Molecular tubes are assembled by stacking cyclic peptides atop one another. The resulting structure defines an interior channel. The diameter of the interior channel is determined by the size of the cyclic peptide, i.e., channel size increase with the size of the cyclic peptide. Cyclic peptides having only 6 amino acid residues have a very small channel suitable for the passage or inclusion of small ions only; cyclic peptides having 16 amino acid residues have a very large channel suitable for the passage or inclusion of small molecules; cyclic peptides having 16 amino acid residues have a very large channel suitable for the passage or inclusion of DNA or RNA.
The repeating D-L-chirality motif is thought to stabilize the disk conformation of cyclic homodetic peptides by lowering the energy of the outwardly oriented conformation of amino acid side chain groups. In the outwardly oriented conformation, side chain groups of amino acid residues are oriented perpendicular to the axis of the disk in a radially outward direction. Orienting the amino acid side chains in this conformation also orients the backbone carboxyl groups and backbone amino hydrogens in a generally axial direction. Orienting the backbone carboxyl groups and backbone amino hydrogens in this axial direction predisposes cyclic peptides to stack atop one another in an anti-parallel fashion so as to form xcex2-sheet hydrogen bonding.
The kinetics of assembly and disassembly of cyclic peptide to form molecular tubes can be controlled by the selection of amino acid side chain groups. Cyclic peptides with ionizable amino acid side chains display pH dependent kinetics. Charged cyclic peptides are found to resist tube assembly; neutralized cyclic peptides are found to promote tube assembly. For example, cyclic peptides incorporating glutamic acid are found to spontaneously assemble into molecular tubes at acidic pH but resist assembly into molecular tubes at alkaline pH. Pre-assembled molecular tubes are found to spontaneously disassemble when the pH is raised from acid pH to alkaline pH. Judicious selection of amino acid side chains can promote packing or aggregation of molecular tubes to form tubular bundles.
Cyclic peptides composed entirely or largely of hydrophobic amino acid residues form molecular tubes within lipid bilayers. Such molecular tubes can span a membrane and provide an ion or molecular channel across such membrane. Such molecular tubes can be employed for loading cells or lipid vesicles with ions or molecules from the extra-vesicular space, depending upon the channel size of the tube. Transmembrane molecular tubes may also be gated so as to control the diffusion of ions and molecules through the channel.
Molecular tubes may be loaded with ionic or molecular inclusions within the channel space. If such tubes are loaded with a drug, the tubes may be employed as a drug delivery system. Release of the drug from molecular tubes may occur by diffusion or by tubular disassembly.
Molecular tubes may also be employed to facilitate the controlled growth of inorganic clusters, semiconductors, and atomic scale wires by means of tube assembly and/or diffusion within the tube channel to produce materials having novel optical and electronic properties.